The goal of the proposal is to identify the factors that limit in vivo survival and function of human ES (hES) derived dopamine (DA) neurons in preclinical models of Parkinson's disease (PD). Preliminary work in our lab has provided high-yield protocols for the derivation of midbrain DA neurons from hES cells. However, pilot transplantation studies of hES cell derived DA neurons in rodent and non-human primates suggest poor in vivo survival and maintenance of DA fate. Poor in vivo survival is in contrast to work with mouse ES derived DA neurons that showed robust survival in both allo- and xenograft PD models. This suggests that hES cell derived DA neurons exhibit a particular vulnerability that needs to be resolved before the clinical use of these cells can be envisaged. Here we propose to systematically address whether cell or host-based parameters or a combination of the two are responsible for the limited in vivo DA neuron survival and function in rodent and monkey models of PD. These studies should provide the basic foundation for our long-term efforts to translate hES cell based DA neuron differentiation strategies for clinical application. The Study has two major aims: 1. To address whether low numbers of surviving DA neurons in vivo are due to incomplete midbrain DA neuron differentiation in vitro 1.1. Improving patterning of hES derived neural rosettes towards midbrain DA neuron identity 1.2. Timing of patterning in neural rosettes: Early vs. late rosettes as a source of DA neurons. 2. To address whether low numbers of surviving DA neurons in vivo are due to selective cell death of the grafted hES-derived DA neuron population. 2.1. Intrinsic vulnerability of hES derived DA neurons 2.2. Lack of sufficient trophic support in vivo 2.3. Host immunological/inflammatory response interferes with DA neuron survival